Studies on Molecular, Phytochemical and Antimicrobial Analysis of Sida Species

97 Pages·2015·1.92 MB·English

Studies on Molecular, Phytochemical and
Antimicrobial Analysis of Sida Species

A Thesis Submitted to the
Orissa University of Agriculture and Technology

In Partial Fulfilment of the Requirement
For the Degree of

Master of Science in Agriculture
(Agricultural Biotechnology)

By
RAJKAMAL SRIVASTAVA
(Adm. No. 05ABT/13)

DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
BHUBANESWAR-751003, ODISHA
2015
ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY
DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
BHUBANESWAR-751003, ODISHA

Dr. Kailash Chandra Samal
Associate Professor Place: Bhubaneswar
Department of Agricultural Biotechnology Date: 23.07.2015

CERTIFICATE-I
This is to certify that the thesis entitled “Studies on Molecular, Phytochemical and
Antimicrobial Analysis of Sida Species” submitted in partial fulfilment of requirements for
the award of the degree of Master of Science in Agriculture (Agricultural Biotechnology)
to the Orissa University of Agriculture and Technology, Bhubaneswar is an authentic record
of bona fide and original research work carried out by Rajkamal Srivastava (Adm. No.
05ABT/13) under my guidance and supervision. No part of this thesis has been submitted for
any other degree or diploma.
It is further certified that the assistance and help received by him from various
sources during the course of investigation has been duly acknowledged.

(KAILASH CHANDRA SAMAL)
Chairman
Advisory Committee

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CERTIFICATE-II

This is to certify that the thesis entitled “Studies on Molecular, Phytochemical and
Antimicrobial Analysis of Sida Species” submitted by Rajkamal Srivastava (Adm. No.
05ABT/13) to the Orissa University of Agriculture and Technology, Bhubaneswar in partial
fulfilment of the requirements for the degree of Master of Science in Agriculture (Agricultural
Biotechnology) has been approved by the students’ advisory committee and the external
examiner.

Advisory Committee

Chairman Dr. K. C. Samal
Associate Professor
Department of Agricultural Biotechnology
College of Agriculture
O.U.A.T., Bhubaneswar ____________________________

Members 1. Prof. G. R. Rout
Professor and Head
Department of Agricultural Biotechnology
College of Agriculture
O.U.A.T., Bhubaneswar ___________________________

2. Dr. Gyanalok Das
Senior Scientist (Horticulture)
High Altitude Research Station, O.U.A.T

Pottangi, Koraput, Odisha ____________________________

EXTERNAL EXAMINER ___________________________

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ACKNOWLEDGEMENT

Mere words are inadequate to express the sense of gratitude and indebtedness to those whose
assistance was indispensable for the completion of my thesis work. But often we have to give vent to our
sentiment through the medium of words written or spoken. First of all, I ascribe all glory to the gracious
Lord Jagannath for the heartfelt benedictions and silent blessings which always act as my savior in the
face of all sorts of peril and paved the path of my life. I have been accompanied and supported by many
people. It is a pleasant aspect that I got a golden opportunity to express my gratitude to all of them.
This perspicuous piece of acknowledgement gives an opportunity and profound privilege to
express my deep sense of gratitude and indebtedness to my adorable guide and chairman of advisory
committee Dr. K. C. Samal, Associate Professor, Department of Agricultural Biotechnology for his
inspiring guidance, valuable suggestion, keen interest, precious advice, constructive criticism and timely
advice during the entire course of investigation and preparation of manuscript. His scientific approach,
innovativeness and generosity without any reservation have my privileges to work under his supervision,
knowledge and enthusiastic interest, which he provided me throughout my post graduation and research
investigation despite his busy schedule of work.
I feel immense pleasure in expressing my heartiest thank and deep sense of gratitude to my co-
advisors Prof. G. R. Rout, Professor and Head, Department of Agricultural Biotechnology and Dr.
Gyanalok Das, Senior Scientist (Horticulture), HARS, Pottangi, OUAT for their enduring interest,
inspiring suggestions to achieve the destination during the entire move despite their heavy work schedule.
I feel proud to convey my heartfelt sense of gratitude to Dr. I. C. Mohanty, Associate Professor,
Department of Agricultural Biotechnology, Dr. A. B. Das, Professor and Head, Department of Botany,
Utkal University and Dr. S. K. Tripathy, Professor, Department of Agricultural Biotechnology for their
regular encouragement, timely advice whenever required during the most trying time in this tenure of
research work.
I am also grateful to Dr. N. Sahoo, Associate Professor and Mr. G. S. Acharya, Technician,
Central Laboratory for their help and guidance in using different equipments during my research
investigations.
Such endeavor is impossible without family support and I was lucky to enjoy this at all stages.
Thus words cannot express my heartiest gratitude for my dearest Mother Kusum Srivastava, Father
Rajesh Kumar Srivastava, Sister Pragati Srivastava, Grandfather Umashankar Lal Srivastava and my
Grandmother.
Two years full of enthusiasm and entertainment, I never think that I will ever be parted from
company of respectable seniors, my friends and loving juniors. It is really heart-rending to depart from
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such happy faces. My words may be not enough to give special thanks to dearest Shonnu, Rahul, Arjun,
Khandoji, Mitali, Vishal, Chetan, Akshay, Binodini, Madhu, Anuradha, Swagatika, Siddarth, Manorama
and Jyotsana who remain very close to my heart for making these two years a wonderful one. This
achievement was not possible without their support and affection.
I am deeply indebted to respected seniors Gaurav Vatsa, Kundan Jadhao, Arundhati Das,
Dhaneswar Swain, Tapaswini Hota, Rinny Swain, Shubhsree Parida, Amrita Priyadarshini, Tanushree
Ghosh, Navnath Munde, Kirath Singh, Sachin Kharade, Saurabh Tripathy, Nihar Ranjan, and Pallavi Jogi
who helped me directly or indirectly to complete this investigation.
I wish to extend my sincere thanks to Pradeep Behera, Amaiya ranjan and other staffs at
Department of Agricultural Biotechnology, College of Agriculture, Bhubaneswar for the help and
assistance.
I have no words to convey to all my well wishers by name, but each of them knows that they
have my cordial thanks.
I am thankful to Department of Biotechnology, Government of India for providing financial
assistance during my entire course of study.
At last I am thankful to those whom have not been mentioned but none is forgotten.

Place: Bhubaneswar Rajkamal Srivastava
Date : 23.07.2015 Adm. No. 05ABT/13

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CONTENTS

PAGE NO.
CHAPTER PARTICULARS
1-8
I INTRODUCTION
9-28
II REVIEW OF LITERATURE
29-42
III MATERIALS AND METHODS
43-62
IV RESULTS
63-68
V DISCUSSION
69-72
VI SUMMARY AND CONCLUSION
73-86
REFERENCES
87-92
APPENDICES

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LIST OF FIGURES

Fig. No. Title Page No.

1 Flow chart summarizing tissue culture experiment 3

2 Chemical structure of cryptolepine 7

3 Selected Sida plant species 44

4 Stages of direct regeneration of Sida cordifolia 48

5 Callus induction for indirect regeneration of Sida
50
cordifolia

6 Individual peaks scanned at 254 nm for cryptolepine 54

Antimicrobial activity of plant extracts on some
7
57
pathogenic bacteria
8 Genomic DNA profile of 4 genotypes of Sida 60

9 Dendrogram depicting genetic relationship among 4
61
genotypes of Sida based on ISSR profile

10 Two dimensional scaling by Principal Component

62
Analysis of 4 genotypes of Sida using Jaccard’s similarity

coefficient of ISSR profile data
11 Three dimensional scaling by Principal Component
Analysis of 4 genotypes of Sida using Jaccard’s similarity

62
coefficient of ISSR profile data

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LIST OF TABLES
Table No. Title Page No.
1 The species of Sida under study and their chemical
24
composition
2 Bacterial strains used in the study 37
3 Morphological characteristics of Sida species 43
Effect of plant growth regulator BAP on direct shoot
4
induction of Sida cordifolia 46
5 Effect of plant growth regulators BAP and NAA on shoot
multiplication of Sida cordifolia 47
Effect of plant growth regulators BAP and NAA on root
6
multiplication of Sida cordifolia 47
7 Effect of plant growth regulators 2,4-D and BAP on callus
induction of Sida cordifolia 49
Extraction of secondary metabolites from leaves of four
8
51
Sida species
9 Peak table of standard cryptolepine and Sida plant samples 53
10 Percentage of cryptolepine in various Sida species 53
Phytochemical evaluation of various leaf extracts of Sida
11
55
species
Antimicrobial activity of crude extracts of Sida species
56
12

DNA profile and polymorphism generated in 4 genotypes
13
61
of Sida using ISSR primer
Similarity coefficient Matrix of 4 genotypes of Sida
14 61
species using ISSR primer
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ABBREVIATIONS
µg : Microgram
µM : Micromolar
2,4-D : 2,4-Dichlorophenoxy Acetic Acid
ANOVA : Analysis of Variance
AU : Arbitrary unit
BAP : Benzyl aminopurine
Bp : Base Pair
cm : Centimetre
D : Day
DW : Dry Weight
EDTA : Ethylene diamine tetra acetic acid
Fig. : Figure
FW : Fresh Weight
G : Gram
Ha : Hectare
HCL : Hydrochloric acid
HPTLC : High performance Thin Layer Chromatography
IAA : Indole-3-acetic acid
IBA : Indole-3- butyric acid
L : Litre
LB : Luria Bertani
M : Molar
Mg : Milligram
mg/l : milligram per Litre
mg/L : milligram per Litre
Min. : Minute
Ml : Millilitre
MS : Murashige and skoogs medium
N : Normal
NAA : α-Napthalene Acetic Acid
NaOH : Sodium hydroxide
nm : Nanometer
OD : Optical density
OUAT : Orissa university of Agriculture and Technology
PCR : Polymerase chain reaction
pH : Potentia Hydrogenii
Ppm : Parts Per Million
Rf : Retention factor
Rpm : Revolution per minute
T : Treatment
TBE : Tris –EDTA
TE : Tris-EDTA
TLC : Thin layer chromatography
v/v : volume/volume
w/v : weight/volume
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CHAPTER- 1

INTRODUCTION

India has rich heritage of use of medicinal plants in clinical practices since ancient times.
In countries like China, Africa, Brazil the medicinal plants are widely used in treatment of
diseases for millenniums. It is known that 80% of world population uses medicinal plants for
various purposes, in traditional system as well as modern medicine (Damintoti et al., 2005). Out
of total medicines prescribed, 30% of herbal medicines are prescribed by physicians. It is
estimated by WHO that most of the population of developing countries uses products of
medicinal plants, for primary health care. In India, various plants are commonly used in disease
treatment of Ayurvedic system. Homeopathic, Siddha, Naturopathy, Unani like alternative
medicine systems also comprise various products obtained from plants. There are thousands of
plant species which may be employed in clinical practices. Various plant parts like roots, stem,
bark, leaves, flowers, fruits, seeds & whole plants are used in various forms of medicines
(Kokate et al,. 2006). They may be used alone or in combination with other plant parts. There are
very clear ancient references for utilization of these plants in clinical treatments. However, there
are not sufficient studies describing the mechanisms correlated to activities of plants. These
studies may lead development of new & cheaper kinds of treatment of many diseases. By using
modern anatomical, phytochemical, biochemical, biotechnological, bioinformatics tools, the
traditionally important molecules may be assessed for their activities.
Sida is one of such ethno medicinally important genus of plants. Sida is
a genus of flowering plants in the mallow family, Malvaceae. They are distributed
in tropical and subtropical regions worldwide, especially in the Americas. Plants of the genus
may be known generally as fanpetals or sidas. The plants from genus Sida are used in India over
2000 years. The plants are also described in various Ayurvedic reference books like Bhav
Prakash Niganthu, Niganthu Ratnakar,Charak Sanhita etc. Various species like Sida cordifolia,
Sida acuta, S. rhombifolia , S. spinosa, S. carpenifolia, S. humilis, S. veronicaefolia are used in
Ayurvedic system. It is used for various purposes like diseases of respiratory system, neural
diseases etc as described in Ayurveda. The plants are also quiet prone for adulteration due to
availability of number of species and also due to lack of sufficient information.
Different species of Sida (and their Sanskrit terminology) that has been taken for the
present study are listed below.
1) Sida cordifolia.L : Bala, Bhadrabala
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